Background: Research has shown that understanding the concepts and principles of health-related fitness is positively related to physical activity; and is vital to becoming physically fit and promote positive attitudes toward fitness. Thus, physical education teachers should be knowledgeable and confident in their own content knowledge of physical activity and health- related fitness in order to provide high quality and effective instruction to their students. This in turn makes it important to investigate the physical activity and health-related fitness content knowledge of inservice physical education teachers. Purpose: The purpose of this study was to examine teachers' content knowledge level on appropriate physical activity and health-related fitness and see how they relate to gender and teaching experience. Methods: Fifty female (n = 31) and nineteen male (n = 19) in-service elementary physical education teachers (ages 23 to 64) from an urban school district in southeast Texas participated in the study. Participants completed an online survey with 41 questions on appropriate physical activity and health-related fitness. Content validity was established through consultation with a group of experts and reliability through administration of a pilot study. Analysis/Results: The survey was found to have a KR20 reliability coefficient of 0.68. Using Spearman-Brown the estimated reliability was .80. The overall mean score on the survey was 55.46%. Females scored slightly higher (M = 56.57%) than male teachers (M = 53.65%). Teachers with less than 6 years of experience scored higher (M = 59.21%) than teachers with 6 to 19 years experience (M = 49.90%), and those with more than 19 years experience (M = 56.91%). None of these differences were statistically significant. A 2 x 3 (Gender x Years of Teaching Experience) ANOVA used to examine the effect of teaching experience and gender on content knowledge found no significant effects on any of the factors. Conclusions: Based on previous studies, the overall mean score (55.46%) for the survey indicated deficiencies on teacher content knowledge, which could relate to their efficacy in achieving the desirable standards. Further, characteristics such teaching experience and gender did not influence appropriate physical activity and health-related fitness knowledge. These findings are consistent with other investigations and suggest the need for on-going and meaningful professional development activities in order to improve the teaching of physical activity and health-related fitness in schools. Keyword(s): exercise/fitness/physical activity, physical education PK-12, professional preparation